This proposal describes a method for automating the Sanger procedure for sequencing polynucleotides. It is based on a mass spectrometric determination of the four component terminal nucleotide residues, where the information regarding the identity of the individual nucleotides is contained in the mass of a stable nuclide marker. Specifically, the four stable isotopes of sulfur (32, 33, 34, 36) will be used to identify the four terminal bases (A, T, G, C). The label will be incorporated as a thiophosphate bridge. The nucleotide fragments will be separated by capillary electrophoresis. The identify of the isotope marker for the terminal base will then be determined by combustion, and mass spectrometric analysis of the resulting isotopically labeled sulfur dioxide. The experimental design aspects of the overall project will include optimization of the capillary electrophoretic separation of nucleotides, development of an electrophoresis-combustion- ionization interface for the mass spectrometer, investigation of the enzymology of the polymerase reaction with thiotriphosphate analogs of natural nucleotides, and an efficient synthesis route to the optically active, isotopically labeled reagents. The method and envisioned instrument can be used to carry out high speed, fully automated DNA sequencing, and should greatly facilitate the national effort to map and sequence the human genome.